FR2668496A1 - L-lysine prodn. useful in animal nutrition - by fermenting new selena:lysine resistant mutants of Brevibacterium or Corynebacterium, providing high yield - Google Patents

Abstract

Prodn. comprises culturing in a nutrient medium, a mutant (L-lysine) (I)-producing strain of Brevibacterium or Corynebacterium which is resistant to selenalysine (II). (I) is accumulated in, then recovered from, the culture broth. The mutant strains are themselves claimed. Culture is pref. carried out at 30-35 deg.C for 40-100 hrs. with pH kept at 6.5-7 during the initial stages and during fermentation. Pref. microorganisms accummulate (I) to at least 2.21 g/dl. Specified are B. lactofermentation AJ 12564 (FERM BP-3554) and AJ12566(BP-3556); C. acetoacidophilum AJ 12565 (BP-3555), B. flavum AJ 12568 (BP-3557) or C. glutamicum AJ 12579 (BP-3559). These strains are prepd. by exposing a starting strain to mutagenic treatment (exposure to UV light or to a chemical mutagen, e.g. N-methyl-N-nitro- N-nitrosoquanidine (NG) or HN03) then selection for resistance to (II) at a concn. which inhibits the starting strain. USE/ADVANTAGE - (I) is an essential amino acid used e.g. in feeding pigs and poultry. It can be now produced more cheaply and efficiently than by known methods.

Description

The present invention relates to a process for the production of

tion of L-lysine by fermentation.

  L-lysine is one of the essential amino acids.

  the first limiting amino acid in cereals and an important amino acid

  so much used in the feeding of roasting chickens or pigs.

  In conventional processes for producing L-lysine, artificial variants of isolated microorganisms derived from the natural world have been used. It is known that most of these artificial variants are derived from microorganisms belonging to the genus Brevibacterium or to the genus Corynebacterium On also knows that the L-lysine biosynthesis system has been potentiated by

  conferring properties on microorganisms such as-

  trophy, resistance as well as sensitivity to analogous amino acids, chemical drugs and so on in combination or successively adding these properties ("AMINO SAN HAKKO (Amino Acid Fermentation)" edited by H Aida, K Takinami, I. Chibata, K Nakayama and H Yamada; Gakkai Shuppan Center, 1986,

page 273.

  However, conventional processes are quite expensive and relatively ineffective, so a process is needed to

  produce L-lysine which is less costly and has an effective

city improved.

  It is therefore an object of the present invention to provide a process for producing L-lysine which is less

  expensive than conventional methods.

  It is also an object of the invention to provide a method for producing L-lysine which is more efficient than conventional methods. The above and other objects are achieved by a process for producing L-lysine, which consists in cultivating in a

  nutrient medium a mutant layer producing L-lysine appears

  linked to the genus Brevibacterium or to the genus Corynebacterium and resistant to selenalysin, to produce and accumulate L-lysine in the culture broth and to collect L-lysine from

culture broth.

  According to the present invention, a method is proposed for

  produce L-lysine by fermentation which is both

  inexpensive and efficient compared to the processes

  to produce L-lysine.

  The Applicant has carried out extensive studies to

  obtain variants or mutants having an L- productivity

  improved lysine from microorganisms belonging to the genus Brevibacterium or to the genus Corynebacterium Consequently, it has surprisingly been discovered that mutant strains having improved L-lysine productivity can be obtained with a high frequency in the variants to which we have confers resistance to seLenalysin The present invention was made on the basis of this discovery Therefore, the present invention provides a method for producing L-lysine which consists in cultivating in a nutritive medium a mutant strain producing L-lysine belonging to the genus Brevibacterium or Corynebacterium and resistant to seLenalysin, to produce and accumulate L-lysine in the culture broth and to collect L-lysine from the broth

of culture.

  The selenalysin (hereinafter abbreviated as SéLys) mentioned in the present invention has a chemical structure similar to that of lysine, in which the methylene group in position y

  lysine is replaced by a selenium (Se) atom.

  Derivative variants used in the present invention (hereinafter abbreviated as parent strains) can be used as mother strains any bacteria whatever the species and the strain as long as they belong to the

  genus Brevibacterium or genus Corynebacterium.

  For example, the following microorganisms known as coryneform bacteria known as L-glutamic acid can be used: Brevibacterium divaricatum ATCC 14020 Brevibacterium flavum ATCC 14067 Brevibacterium lactofermentum ATCC 13869 Brevibacterium roseum ATCC 13825 Corynebacterium acetoacidophilum ATCC 13870 Coryneumumoryumum ATCC 13032 In addition, it is also possible to use strains producing L-lysine which are obtained by conferring on microorganisms

  described above properties such as auxotrophy at L-

  alanine and sensitivity to fluoropyruvate, for example, which are

  already known to be effective in improving the productivity of L-

  lysine. The resistance to Sélys mentioned in the present invention is understood to designate the property of such a variant

  which can grow in an environment containing Sélys at a concentration

  tration which inhibits the growth of the mother strain.

  The variants used in the present invention can be easily obtained by subjecting the mother strains described above to a mutagenic treatment such as, for example, exposure to UV or treatment with chemical agents such as Nmethyl-N'-nitro -N-nitrosoguanidine (hereinafter NG) or nitric acid and then collecting the variants which

  can grow in a medium containing Sélys at a concentration

  tration, for example 500 pg / ml, which inhibits the growth of

mother strain.

  Specific examples of the variants which can be used in the present invention include the following strains: Brevibacterium lactofermentum AJ 12564, FERM BP-3554 (Sélysr) Corynebacterium acetoacidophilum AJ 12565, FERM BP-3555 (Sélys) Brevibacterium lactofermentum AJ 12566, FERM BP- 3556 (AEC, Ala, Sélysr) Brevibacterium flavum AJ 12568, FERM BP-3557 (AEC r, PK W Sélysr) Corynebacterium glutamicum AJ 12579, FERM BP-3559 (AEC r Sélysr) The symbols used in parentheses have the following meanings: Selysr : resistance to selenalysin AEC: resistance to S- (2-aminoethyl) -cysteine Ala: auxotrophy with alanine

  P Kw: reduced activity of pyruvate kinase.

  The variants described above were deposited on October 1990 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (FRI), 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan and then transformed on September 10, 1991 into a

  deposit under the Budapest Treaty.

  As sources of carbon in the nutrient medium which is used to cultivate the present mutant strain, sugars such as glucose and molasses can be used, for example; organic acids such as, for example, acetic acid and acid

  citric; and alcohols such as, for example, ethanol.

  As nitrogen sources, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, urea, aqueous ammonia and gas can be used.

  ammonia and other conventional sources of nitrogen.

  As organic nutrients, there can be used, for example, soybean acid hydrolyzate and yeast extract. The conditions for the culture of the mutant strain according to

  the invention are preferably aerobic conditions at a temperature

  Fermentation rate of approximately 30 to 35 ° C. for a fermentation period of approximately 40 to 100 h. It is preferred to maintain the p H in the range of approximately 6.5 to 7.0 in the initial stage and during the fermentation To adjust the p H, it is possible to use conventional agents such as inorganic or organic acid or alkaline substances and in addition urea, carbonate of

calcium or ammonia gas.

  L-lysine can be collected from the fermented broth.

  tion in general by the ion exchange resin process and

  other techniques known in combination.

  The present invention will be illustrated below by

some nonlimiting examples.

Example 1

  Brevibacterium lactofermentum ATCC 13869 was cultured in a medium containing 1% peptone, 1% yeast extract, 0.5% Na Cl and 0.5% glucose, the p H being adjusted to 7.0 After having collected the cells, treatment was carried out with 0.1 M phosphate buffer (p H 7.0) containing 250 μg / ml of NG at 30 ° C. for 30 min Then the cells having a survival rate of 1.0 % were inoculated on a minimum medium on an agar plate containing 500 μg / ml of Selys (table 1) and cultured at 30 ° C. for 7 days. It was obtained from the colonies which grew

  Brevibacterium lactofermentum AJ 12564 (FERM BP-3554).

  Brevibacterium lactofermentum ATCC 13869 which is the strain

  mother cannot grow (sensitive to Sélys) on the mini environment

  mum on agar plate above containing 500 μg / ml of Sélys, while the variant obtained, Brevibacterium lactofermentum AJ 12564, can grow on the medium (resistant to Sélys) The variant used in the present invention can be clearly

  distinguished from the mother strain or other variants by this difference

rence.

Table 1

  Composition of the minimum medium Component Concentration Glucose 20 g / l Ammonium sulphate 10 g / l KH 2 PO 4 1 g / l Mg SO 4.7 H 20 0.4 g / l Fe SO 4.7 H 20 10 mg / l Mn SO 4.4 H 20 10 mg / l Biotin 50 pg / l Thiamine hydrochloride 100 pg / l Urea 2 g / l (p H 7.0)

Table 2

  L-lysine production medium Component Concentration Glucose 100 g / l Ammonium sulphate 45 g / l KH 2 PO 4 1 g / l Mg SO 4.7 H 20 0.4 g / L Fe SO 4.7 H 20 10 mg / L Mn SO 4.4 H 20 10 mg / l Biotin 50 pg / l Thiamine hydrochloride 200 pg / l Soy protein hydrolyzate concentrate (total nitrogen 7%) 15 pg / l (p H 7, 0) Next, for the production of L-lysine by fermentation, the L-lysine production medium indicated in Table 2 was prepared and two samples of 20 ml of the medium were each loaded separately into a shaken flask of a volume of 500 ml After sterilization by heating at 115 ° C. for 10 min, 1 g of calcium carbonate previously sterilized with dry air was added to the medium. Brevibacterium lactofermetum ATCC 13869 and AJ 12564 were inoculated on the medium, respectively. then cultured at 31.5 C for 72 h on a shaker The amount of L-lysine (calculated as L-lysine monohydrochloride) accumulated in the fermentation broth was quantitatively determined and is indicated in Table 3 with the resistant mutant strain to Sélys, a net increase is observed in the production of

  L-lysine in comparison with the mother strain.

Tab 7 water 3

Table 3

  Strain Sensitivity L-lysine, HCL to accumulated Sélys (g / dl) ATCC 13869 sensitive 0.01 AJ 12564 resistant 2.32 Example 2 Corynebacterium acetoacidophilum ATCC 13870 was mutated by treatment with NG in a similar manner to example 1 to give Corynebacterium acetoacidophilum AJ 12565 (FERM BP-3555) as a strain resistant to Sélys Corynebacterium acetoacidophilum ATCC 13870 and AJ 12565 were cultivated in the same way

  so that in Example 1 and the production of L-lysine was studied.

  The results are shown in Table 4.

Table 4

  Strain Sensitivity L-lysine, HCL to accumulated Sélys Lé (g / dl) ATCC 13870 sensitive 0.01 AJ 12565 resistant 2.21

Example 3

  Brevibacterium lactofermentum AJ 3424 (FERM P-1711) resistant to S- (2-aminoethyl) -L-cysteine and auxotrophic for alanine has been subjected to mutagenic treatment with NG in a similar way to Example 1 to give Brevibacterium lactofermentum AJ 12566 (FERM BP-3556) as a strain resistant to Sélys Brevibacterium lactofermentum AJ 3424 and AJ 12566 were cultivated in the same way as in Example 1 and the production of L was studied -lysine The results are shown in the table. The quantity produced was improved by 20.3% with

  Brevibacterium lactofermentum AJ 12566 in comparison with AJ 3445.

Table 5

  Strain Sensitivity L-lysine, HCL accumulated SéLys (g / dl) ATCC 3424 sensitive 2.32 AJ 12566 resistant 2.79

Example 4

  Corynebacterium glutamicum AJ 3463 (FERM P-1987) producer

  Sys (2-aminoethyl) -L-cycteine-resistant lysine was subjected to mutagenic treatment in a manner similar to Example 1 to give Corynebacterium glutamicum AJ 12579 (FERM BP-3559) as a strain resistant to Selys Corynebacterium glutamicum AJ 3463 and AJ 12579 were cultivated in the same way as in Example 1 and the production of L-lysine was studied. The results are

shown in Table 6.

Table 6

  Strain Sensitivity L-lysine, HCI to accumulated Sélys (g / dl) ATCC 3463 sensitive 2.21 AJ 12579 resistant 2.64

Example 5

  Brevibacterium flavum ATCC 11841 (FERM P-6463) resistant to S- (2-aminoethyl) -L-cysteine, whose pyruvate kinase activity had been reduced in comparison with the wild-type strain, was subjected to mutagenic treatment similar to Example 1 to give Brevibacterium flavum AJ 12568 (FERM BP-3559) as a strain resistant to Sélys Brevibacterium flavum AJ 11841 and ATCC 12568 were cultivated in the same way as in Example 1 and studied the production of L-lysine Les

  results are shown in Table 7.

Table 7

  Strain Sensitivity L-lysine, H Cl to accumulated Sélys Lé (g / dl) ATCC 11841 sensitive 4.00 AJ 12568 resistant 4.50

Example 6

  Each time with a platinum loop, Brevibacterium flavum AJ 11841 and ATCC 12568, a culture was taken on an agar declined and was inoculated on 50 ml of culture medium for seeding indicated in Table 8, respectively. The broth was prepared. inoculation culture by carrying out the agitated aerial culture at 31.5 C for 18 h. On the other hand, 300 ml of the main culture medium indicated in Table 9 were loaded separately into a 1 l glass fermenter and each time inoculated with 15 ml of the culture seed broth. air culture stirred at 31.5 C with aeration at an hourly space velocity of -1 1 min 1 While maintaining the p H of the medium in the range of 7.0 to 8.0, was added continuously or by intermittent in the culture broth, a mixture of glucose and ammonium sulphate (ratio of glucose / ammonium sulphate mixture 10: 1; concentration of glucose in the mixture 40%) to carry out the fermentation at 31.5 C for 72 h The results are shown in Table 10.

Table 8

  Composition of the seeding culture medium Component Concentration Glucose 15 g / l Ammonium sulphate 3 g / l Urea 1 g / l KH 2 PO 4 1 g / l Mg SOL ', 7 H 20 0.4 g / l Mn 2 mg / l ++ Fe 2 mg / L Biotin 50 pg / L Thiamine hydrochloride 200 pg / l Soy protein hydrolyzate concentrate (total nitrogen 7%) 30, pg / l (p H 7.0)

Table 9

  Composition of the main culture medium Component Concentration Glucose 10 g / l Ammonium sulphate 30 g / l KH 2 PO 4 1 g / l Mg SO 4, 7 H 20 0.4 g / l ++ Fe 2 mg / L ++ Mn 2 mg / l Biotin 50 pg / l Thiamine hydrochloride 60 pg / l Soy protein hydrolyzate concentrate (total nitrogen 7%) 30 pg / l (w H 7.5)

Table 10

  Strain Sensitivity L-lysine, HCI to Sélys accumulated (g / dl) ATCC 11841 sensitive 8.60 AJ 12568 resistant 9.50 By thus cultivating the strain resistant to Sélys in a nutritive medium, the L-lysine accumulated in the broth of culture increases markedly in comparison with that accumulated by the mother strain. Consequently, the cost of production of L-lysine by fermentation is greatly reduced, in particular at

industrial scale.

  It is understood that the invention is not limited to

  preferred embodiments described above by way of illustration

  tion and that a person skilled in the art can make modifications to it

  without departing from the scope of the invention.

Claims (10)

  1 Process for the production of L-lysine, characterized in that it consists in cultivating in a nutritive medium a mutant strain producing L-lysine belonging to the genus Brevibacterium or to the genus Corynebacterium and resistant to selenalysin, to produce and accumulate the L-lysine in culture broth and   collect the L-lysine from the culture broth.   2 The method of claim 1, further characterized in that said strain is grown in the culture medium at a fermentation temperature of about 30 to 35 C for about 40 to 100 h. 3 The method of claim 2, further characterized in that the p H is maintained at about 6.5 to 7.0 in the step   initial and during fermentation.   4 Method according to claim 1, characterized in that said strain belonging to the genus Corynebacterium or to the genus Brevibacterium and resistant to selenalysin is chosen from   Brevibacterium lactofermentum AJ 12564, Corynebacterium acetoacido-   philum AJ 12565, Brevibacterium lactofermentum AJ 12566, Brevibac-   terium flavum AJ 12568 and Corynebacterium glutamicum AJ 12579.   Process according to which L-lysine is produced 6 Process according to L-lysine is produced 7 Process according to L-lysine is produced 8 Process according to L-lysine is produced 9 Process according to L-lysine is produced Process for claim in quantity of in claim in quantity of in claim in quantity of in claim in quantity of in claim in quantity of at least 1, characterized in minus 2.21 g / dl. 1, characterized by minus 2.31 g / dl. 1, characterized by minus 2.64 g / dl. 1, characterized by minus 2.79 g / dl. 1, characterized by minus 9.50 g / dl.   produce a mutant strain producing L-lysine from a microorganism belonging to the genus bacterium or to the genus Corynebacterium and resistant to nalysin, characterized in that a mother strain is subjected to the genus Brevibacterium or the genus Corynebacterium ce ce this what this Brevi- selected- appar-   mutagenic treatment and variants which are capable of growing in a medium containing selenalysin at a concentration sufficient to inhibit the growth of the mother strain are collected. 11 Method according to claim 10, characterized in that   that said mutagenic treatment includes exposure to ultra-   purple or treatment with a mutagenic chemical compound.   12 Method according to claim 11, characterized in that   that said mutagenic chemical compound comprises N-methyl-N-nitro-N-   nitrosoguanidine or nitric acid.   13 Method according to claim 11, characterized in that   that said mother strain is chosen from Brevibacterium divari-   catum ATCC 14020, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and Brevibacterium roseum ATCC 13825, Corynebacterium lilium ATCC 15990 and Corynebacterium glutamicum ATCC 13032.   14 Mutant strain of microorganism producing   L-lysine characterized in that it belongs to the genus Brevi-   bacterium or genus Corynebacterium, and in that it is resistant to selenalysin.   Mutant strain of L-lysine-producing microorganism according to claim 14, characterized in that it is   chosen from Brevibacterium lactofermentum AJ 12564, Corynebacte-   rium acetoacidophilum AJ 12565, Brevibacterium lactofermentum AJ 12566, Brevibacterium flavum AJ 12568 and Corynebacterium glutamicum AJ 12579.   16 Mutant strain of L- producing microorganism   lysine according to claim 14, characterized in that it   produces L-lysine in an amount of at least 2.21 g / dl.   17 Mutant strain of L- producing microorganism   lysine according to claim 14, characterized in that it   produces L-lysine in an amount of at least 2.31 g / dl.   18 Mutant strain of L-producing microorganism   lysine according to claim 14, characterized in that it   produces L-lysine in an amount of at least 2.64 g / dl. lysine product lysine product   19 Mutant strain of L- producing microorganism   according to claim 14, characterized in that it   L-lysine in an amount of at least 2.79 g / dl.   Mutant strain of L- producing microorganism   according to claim 14, characterized in that it   L-lysine in an amount of at least 9.50 g / dl.